MODULATION OF CALCIUM CONTROL OF CARDIAC MYOFIBRILS

心脏肌原纤维钙控制的调节

• 批准号: 3336761
• 负责人: R John Solaro
• 金额: $ 16.64万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3336761
• 关键词: acid base balance; acidity /alkalinity; actins; adenosine diphosphate; adenosinetriphosphatase; affinity chromatography; calcium binding protein; calcium metabolism; calcium transporting ATPase; cations; ethylene glycols; homeostasis; hydrogen; hypoxia; ion transport; ischemia; laboratory rat; lactates; magnesium; membrane permeability; muscle relaxants; muscle stimulant; muscle tension; myocardium; myofibrils; myosins; nucleotides; phosphorylation; stretch reflex; troponin

The general goals of the proposed research has been to understand the process by which Ca2+ activates the mechanical and biochemical activity of cardiac myofilaments, and to known whether, how, and when this process is modified physiologically by intrinsic mechanisms and by mechanisms extrinsic to the myofilaments involving protein phosphorylation. Experiments relating to intrinsic regulation are aimed at determination of whether Ca2+ binding to regulatory sites in the myofilament lattice are influenced by the sarcomere length and/or by force-generating cross-bridge complexes with the thin filament. Experiments relating to extrinsic regulation focus on phosphorylation of troponin I (TnI), the P-light chain of myosin, and C-protein. The aims are to determine the effect of TnI phosphorylation on the binding of cross-bridges to the thin filament, and also to determine whether there are isoforms of TnI and how variations in the population of the TnI isoforms affect myofilament activity and regulation. In the case of studies on C-protein and the P-light chain of myosin, the aims are to determine whether these proteins affect myofilament activation by Ca2+ and whether their activity is influenced by the level of protein phosphorylation. The approach to these aims involves measurement of levels of covalent phosphorylation of myofilament proteins in hearts freeze-clamped during the response to inotropic perturbations. The approach also involves preparation of each of the main myofilament proteins, analysis of the isoforms of TnI and myosin heavy chain, reconstitution with variations in the levels of covalent phosphorylation and measurement of cross-bridge binding to actin and actomyosin ATPase activity. Mechanical studies using chemically skinned fibers include measurements of tension, stiffness, Vmax, and kinetics of the rise and fall of force during "jumps" and "dives" in the free Ca2+ surrounding the myofilaments. Methods also include newly developed procedures for making multiple measurements of Ca2+ binding to chemically skinned preparations of heart muscle, characterized for TnC content, sarcomere length and extent of actin-cross-bridge interaction. The proposed experiments bear directly on current perceptions of the regulation of cardiac output by the Starling mechanism and by the autonomic nervous system, and whether extrinsic and intrinsic regulation of the Ca2+ activation of the myofilaments might represent a locus of lesions in heart failure.

拟议研究的总体目标是了解 Ca2+ 激活机械和生化活性的过程 心肌丝，并了解该过程是否、如何以及何时进行 通过内在机制和机制进行生理改变 涉及蛋白质磷酸化的肌丝外源性。 与内在调节相关的实验旨在确定 Ca2+ 是否与肌丝晶格中的调节位点结合 受肌节长度和/或产生力的跨桥影响 与细丝形成复合物。 与外源性相关的实验 调节重点是肌钙蛋白 I (TnI)（P 轻链）的磷酸化 肌球蛋白和C蛋白。 目的是确定 TnI 的效果 横桥与细丝结合的磷酸化，以及 还可以确定是否存在 TnI 亚型以及 TnI 亚型的变异情况 TnI 同种型的数量影响肌丝活性 规定。 以C-蛋白和P-轻链的研究为例 肌球蛋白，目的是确定这些蛋白质是否影响肌丝 Ca2+ 的激活以及它们的活性是否受 Ca2+ 水平的影响 蛋白质磷酸化。 实现这些目标的方法涉及测量 心脏中肌丝蛋白的共价磷酸化水平 在对正性肌力扰动的反应过程中被冻结。 这 该方法还涉及准备每个主要肌丝 蛋白质、TnI 和肌球蛋白重链同种型分析， 共价磷酸化水平变化的重构 以及与肌动蛋白和肌动球蛋白 ATP 酶的跨桥结合的测量 活动。 使用化学结皮纤维的机械研究包括 张力、刚度、Vmax 以及上升和下降动力学的测量 在“跳跃”和“潜水”期间，在自由 Ca2+ 周围的力 肌丝。 方法还包括新开发的制作程序 多次测量 Ca2+ 与化学去皮制剂的结合 心肌，以 TnC 含量、肌节长度和范围为特征 肌动蛋白-跨桥相互作用。 所提出的实验直接关系到 目前对椋鸟调节心输出量的看法 机制和自主神经系统，以及是否外在和 肌丝 Ca2+ 激活的内在调节可能 代表心力衰竭的病变部位。